SUMMARY OF WORK The new 3D model of stochastic calcium release in sino-atrial node cells (SANC) has been elaborated and revised to provide extensive imaging and movie output, and to allow simulations of indefinite length in order to study rhythm effects. An attempt was made to base the model geometry directly on observed immunofluorescence images of RyR distribution, but it was found that the information in conventional light microscopic images has insufficient resolution to define the local pathways by which CICR spreads between neighboring couplons. We have begun to obtain super-resolution microscopy of these cells in collaboration with a microscopy group in NIBIB. Preliminary collaborative studies have confirmed the model result that calcium release events are confined to a thin layer beneath the sarcolemma, which would not be demonstrated with standard confocal microscopy. We have also established a collaboration with Clara Franzini-Armstrong to study structure of SANC by electron microscopy. Extensive statistical results on the distribution of couplons by EM have just arrived and are awaiting analysis. We have developed specialized software to identify and track calcium release events in SANC, and to study their statistics, leading to the discovery that propagated calcium events are important even in early diastole. The model is being extensively exercised to understand the range of conditions in which the coupled-clock mechanism is viable. Further studies have been directed to a pathological regime discovered by modeling, in which the regulatory effects of the adrenergic system on heart rate are reversed when RyR sensitivity is too high. We have begun exploring this regime -- of possible clinical significance in heart failure and genetic abnormalities of RyR and CASQ -- using caffeine to modulate the RyR. Initial results show that, in order to understand arrhythmic beating of SANC in the coupled-clock regime, it will be necessary to improve on the underlying electrophysiological model of these cells that has been widely accepted. We have begun electrophysiological studies of SANC with that goal. We are also using the 3D model to study the mechanism of heart rate variability at the single-cell level, which requires simulations lasting for days.